拟南芥F-box 蛋白XS01C 参与气孔发育泛素化调控途径的研究

Stomata are plant-specific epidermal structures which play important function in gas exchanges requiring for the process of photosynthesis and water evaporation. Arabidopsis FOUR LIPS (FLP) is the first identified key MYB transcription factor regulating the terminal cell division and differentiation during stomatal development. By means of screening of EMS-mutated flp-1 mutants, we found that flp-1 xs01c mutant displays an aggressive stomatal defective phenotype in comparing with flp-1 single mutant, leading to excessive cell divisions and retarded cell differentiation. Map-based cloning analysis indicates that XS01C encodes an F-box protein, a putative component of SCF complexes, however whether the ubiquitination regulatory pathway plays a role during stomatal development has not been reported. Using approaches of molecular, biochemical and cellular biology, we will probe the regulatory network between XS01C and other key factors that function at the terminal stages of stomatal development, identify the involvement of protein ubiquitination pathway during stomatal development. The results will provide new evidences in revealing the mechanism of controlling of cell division and differentiation during plant development.

气孔是植物表皮上重要的特化细胞结构，调控着植物光合作用和蒸腾作用所需气体交换和水分蒸发。拟南芥FOUR LIPS（FLP）是第一个被鉴定的调控气孔发育后期细胞分裂和分化的关键MYB转录因子。我们在前期flp-1突变体EMS诱变突变体筛选中发现，flp-1 xs01c突变体表现出比flp-1明显加剧的异常气孔表型,导致表皮细胞冗余分裂、细胞分化受阻。图位克隆结果发现XS01C编码一个F-box蛋白，但是否作为SCF复合体的组成部分并通过泛素降解途径参与气孔发育还没有报道。本项目中拟利用分子生物学、生物化学和细胞生物学等技术，探讨XS01C与相应气孔发育后期的关键调控因子的网络关系，验证蛋白泛素化也是气孔发育重要调控途径之一，研究结果为深入理解植物发育中细胞分裂和分化调控机制提供新依据。

气孔是分布在所有陆地植物地上器官表面的特化表皮细胞结构调控植物与外界之间气体交换。近年研究结果表明，气孔发育是研究植物细胞分化和分裂的理想系统。在拟南芥气孔发育后期， R2R3-MYB转录因子FOUR LIPS (FLP/MYB124)和MYB88通过对多个细胞周期基因CYCA2;3、CDKB1;1、CDKA;1转录调控抑制保卫细胞母细胞发生冗余分裂。我们从flp-1突变体的EMS诱变群中筛选到一个能明显增强flp-1气孔簇表型的突变体xs01c。XS01C基因编码F-BOX STRESS INDUCED 4 （FBS4）， 特异地在气孔前体细胞内表达。FBS4过表达可以挽救了flp-1 xs01c和flp-1突变体气孔表型的缺陷。FBS4蛋白 F-box结构域内的第166个脯氨酸的缺失或替代影响FBS4与Skp1/Cullin/F-box (SCF) E3泛素连接酶复合体的核心亚基 ASK1相互作用。FBS4蛋白能够与CYCA2;3发生物理相互作用，并通过26S泛素蛋白酶体途径诱导CYCA2;3降解。研究结果表明，除了已知的转录途径外，气孔发育中可以通过F-box蛋白FBS4识别并泛素化靶蛋白CYCA2;3，在翻译后水平上调控对称分裂，为揭示气孔发育中细胞分裂精细调控提供了新的线索。